Human chromosomal DNA have double-stranded DNA with repeated sequences of 5′-TTAGGG-3′ at the terminus, which are called telomeres. However, the terminal of the telomere has a structure in which the 3″-terminal is an overhang, and a single-stranded DNA region of 75 to 300 bases (G tail, hereinafter referred to simply as G tail). The G tail is normally in a protected state, as a loop is formed, except during the access of a telomere-elongating enzyme telomerase or during the replication of DNA (see, for example, Nonpatent Document 1).
A telomere double-stranded region occupying the most of the telomere is known to be shortened after each cell division and thus involved in cell aging, but the G tail retains a certain length of 75 to 300 bases even after repeated cell division. Contradictorily, there are reports showing that a G tail retains a certain length of 75 to 300 bases after the termination of cell division by the shortening of a telomere double-stranded region after many cell divisions, i.e., after the limited replicative senescence, and some other reports showed that a G tail is shortened after the limited replicative senescence. This is probably because there was no method of measuring the length of a G tail accurately and quantitatively, as the G tail is much shorter than the telomere.
On the other hand, the recent discovery of a POT1 protein binding to a G tail but not to a double-stranded telomere DNA, a PIP1 protein binding to the protein, and the like, has showed that a telomere G tail has a function completely different from that of a double-stranded region, i.e., it is involved in direct signaling of cell death, various cell responses, and the like, as described below.
A telomere has telomere-binding proteins binding to the telomere; TRF1 (Telomere repeat binding factor) and TRF2 are known as such telomere-binding proteins; and it has been recently found that cancer cells do not form a G tail loop in the absence of TRF2 and consequently have shortened G tails (see, for example, Nonpatent Document 2). In such case, what is important is that the G tail is shortened, although the entire telomere length remains unchanged and also fused with the chromosome terminal.
In the case of a normal cell, the elimination of function of TRF2 in the cell leads to the shortening of the G tail, the termination of cell proliferation, and consequently to aging (see, for example, Nonpatent Document 2). In this case too, the entire telomere length remains unchanged, suggesting that the shortening of the G tail triggers aging.
Various proteins such as TRF1 and TRF2 described above, as well as ATM, NBS1, and MRN are known to be essential for the formation of a G tail loop. DNA damage-sensitive signals, e.g., caused for example by various DNA damaging agents or radiation, do not trigger the shortening of the telomere, but induce the shortening of the G tail. This is apparent, since proteins needed for DNA restoration (such as ATM, NBS1 and MRN) are recruited.
ATM is a gene responsible for angiectatic diseases, and NBS1 is a gene responsible for Nijmegen syndromes, i.e., a rare autosomal recessive genetic disease characterized by its high carcinogenicity, immunodeficiency, chromosomal instability, and radiosensitivity. Therefore, the recruitment of these genes to the G tail suggests some relationship of the G tail with the above-described diseases. Actually, the inhibition of the function of TRF2 as the adhesive of a G tail loop induces ATM-dependent apoptosis (see, for example, Nonpatent Document 3).
It has been found that the anticancer agents specifically-acting on a G tail lead to the shortening of the G tail without the shortening of the telomere and consequently to the death of cancer cells (see, for example, Nonpatent Document 4).
These results suggest that medicines and stresses causing DNA damage transmit signals to cells via a G tail, causing various cell responses.
In addition, a cancer-inhibiting gene product p53, of which many variants are observed in many cancers, is known to bind to a G tail (see, for example, Nonpatent Document 5), evidently indicating that the change in the G tail is a signal even in diseases associated with cancers and aging.
Since then, there are developed methods of measuring the length of a G tail, including T-OLA (telomere-oligonucleotide-ligation assay), PENT (primer-extension/nick translation), 3′-overhang protection assay, and the like (see, for example, Nonpatent Documents 6 and 7).
However, as will be described below with reference to Table 1, all these methods demand autoradiography and gel preparation with a radioactive label (such as 32P), which are troublesome in handling. Also, electrophoresis demands an elongated period for phoretic separation. For these reasons, all these methods are tedious assays demanding at least two days for completion, which are unfavorable for the real-time monitoring of progress of cancer and rapid diagnosis of clinical outcome. In addition, these methods could not be applied easily to the high-throughput screening for analyzing a great number of samples.
Further, in conventional hybridization protection assay (HPA; see, for example, Patent Document 1 and Nonpatent Document 8), which uses a chromosomal DNA after denaturation, the G tail length, which is approximately 1/100 or less of the entire telomere length, is within the range of its operation and measurement errors, and therefore cannot be measured.
Specifically, because the signal intensity of a G tail obtained by the method is so low at the noise level, it is difficult to determine the G tail quantitatively and accurately and also to identify whether the signal is specific to the G tail.    Patent Document 1: Japanese Unexamined Patent Publication No. 2001-95586    Nonpatent Document 1: Griffith J D, Comeau L, Rosenfield S, Stansel R M, Bianchi A, Moss H and de Lange T., Cell: 97 (1999), 503-14.    Nonpatent Document 2: van Steensel B, Smogorzewska A and de Lange T., Cell: 92 (1998), 401-13.    Nonpatent Document 3: Karlseder J, Broccoli D, Dai Y, Hardy S and de Lange T., Science: 283 (1999), 1321-5.    Nonpatent Document 4: Gomez D, Paterski R, Lemarteleur T, Shin-Ya K, Mergny J L and Riou J F. J. Biol. Chem.: 279 (2004), 41487-94.    Nonpatent Document 5: Stansel R M, Subramanian D and Griffith J D., J. Biol. Chem.: 277 (2002), 11625-8.    Nonpatent Document 6: Chai, W., Shay, J. W. & Wright, W. E., Mol. Cell. Biol.: 25, 2158-2168 (2005).    Nonpatent Document 7: Saldanha, S. N., Andrews, L. G. & Tollefsbol, T. O., Eur. J. Biochem.: 270, 389-403 (2003).    Nonpatent Document 8: Nakamura, Y. et al., Clin. Chem.: 45, 1718-1724 (1999).